Glycogen phosphorylase (a-l,4-glucan:orthophosphate glueosyltransferase, EC 2.4.1.1) may be assayed in either the direction of glycogen synthesis or the direction of glycogen phosphorolysis. In the forward direction, phosphorolysis, progress of the reaction can be monitored continuously or as a fixed-time point speetrophotometrieally (1) or fluorometrieally (2) through the use of coupled enzyme systems involving phosphoglueomutase and glueose-6-phosphate dehydrogenase. A recent report (3) has presented a radioactive method for measurement of the forward reaction in which a2Pi incorporation into glucose 1-phosphate-32P may be counted as a direct measure of activity.
All present assays using the backward reaction ultimately involve the determination of inorganic phosphate produced during glycogen synthesis from glucose 1-phosphate, a method originally employed by Cori and Green (4) and subsequently by Cori (5), and Illingsworth and Cori (6). Conditions of assay have been modified for special purposes such as Hedriek and Fiseher's modification (7) to achieve zero-order kinetics.
Certain of the assays now employed have special advantages particularly with respect to continuous monitoring of the reaction. All of the methods, however, suffer from being either too cumbersome or time consuming. The assay presented in this paper is extremely rapid and, although not designed for continuous monitoring, it is suitable for initial velocity measurements. An additional advantage of the present method is the potential for increased sensitivity of the assay, making it suitable for the development of mieroassays. The procedure employs the principle of the filter paper assay used by Bollum (9) for the study of DNA synthesis and is an adaptation of the methodology used in the glycogen synthetase assay by Thomas et al. (8). A related procedure for the assay of amylo-l,6-glucosidase has been reported (10). In the present method radioactive glycogen is deposited on filter paper squares, washed, and counted. In the present adaptation for phosphorylase assay the 14C-gly-20